Quantitative aspects of active transport by the lactose transport system of Escherichia coli

Accumulation by the lactose transport system of Escherichia coli has been measured in cells induced so as to contain increasing numbers of membrane carriers. Carrier activity was assayed both by the rate of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ entry and the initial rate of accumulation of $\text{methyl-1-thio-gb-}\text{d}\text{-galactopyranoside}$. At the steady-state cells with a low number of carriers accumulated considerable amounts of substrate when compared to the fully induced control. This is consistent with the hypothesis that there are two distinct routes of both entry and of exit: a carrier mediated pathway and a diffusion component. When these two factors are evaluated quantitatively they account for the observed relationship between the number of carriers and the steady-state accumulation achieved.     

Metabolic control of lactose entry in Escherichia coli

A general method has been developed for determining the rate of entry of lactose into cells of Escherichia coli that contain β-galactosidase. Lactose entry is measured by either the glucose or galactose released after lactose hydrolysis. Since lactose is hydrolyzed by β-galactosidase as soon as it enters the cell, this assay measures the activity of the lactose transport system with respect to the translocation step. Using assays of glucose release, lactose entry was studied in strain GN2, which does not phosphorylate glucose. Lactose entry was stimulated 3-fold when cells were also presented with readily metabolizable substrates. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ (ONPG) was only slightly elevated (1.5-fold) under the same conditions. The effects of arsenate treatment and anaerobiosis suggest that lactose entry may be limited by the need for reextrusion of protons which enter during H⁺/sugar cotransport. Entry of $\text{o-}\text{nitrophenyl-β-}\text{d}\text{-galactopyranoside}$ is less dependent on the need for proton reextrusion, probably because the stoichiometry of H⁺/substrate cotransport is greater for lactose than for ONPG.     

Inhibition of growth of Escherichia coli by lactose and other galactosides

A study has been made of the inhibition of growth caused by the addition of lactose or other galactosides to lac constitutive Escherichia coli growing in glycerol minimal medium. The effect was greater at pH 5.9 and pH 7.9 than at pH 7.0. Inhibition of growth by lactose was observed also in the case of a β-galactosidase negative mutant. However, a lacY mutant, which has a defect in the entry of protons normally coupled with galactoside transport, showed only slight inhibition of growth on the addition of galactosides. In the case of the parental strain the addition of lactose resulted in a sharp fall in ΔpH across the cell membrane and a reduction in intracellular ATP, and the recovery was slow. Under the same conditions the lacY mutant showed a smaller and only transient effect. It is postulated that the sudden entry of protons associated with lactose uptake lowers the protonmotive force, reducing the ATP levels and inhibiting growth of the cells. This hypothesis would account also for the selection of lacY mutants found when E. coli is grown in the presence of isopropyl-β-d-thiogalactoside.     

Counter-transport mediated by the lactose permease of Escherichia coli

When the two main energy yielding pathways, respiration and the membrane ATPase of Escherichia coli are poisoned, the lactose permease is unable to accomplish accumulative transport of thiogalactosides, but the efflux of pre-loaded substrate can be coupled to a transiently uphill transport of exogenous substrate. This transient uphill transport, called overshoot has been reexamined with the possibility of an obligate H⁺ cotransport in mind. Overshoot can be diminished but not suppressed by a proton-conducting uncoupler, carbonyl cyanide $\text{m}$ chlorophenylhydrazone, (CCCP) and by a liposoluble cation, triphenyl-methyl phosphonium (TPMP⁺). The effect of other factors, such as temperature, amount of permease and pH were also explored. The overshoot was found to decrease with increasing pH, until at pH 8 it became negligible. This is in sharp contrast with the relatively flat pH dependence of uphill and downhill transport in unpoisoned cells. CCCP and TPMP⁺ had no inhibitory effect on the overshoot at pH 6 and below.